Renal cell carcinoma (RCC, kidney cancer) is one of the most commonly diagnosed cancers and is associated with significant mortality. In the United States in 2011, it is estimated that 60,920 patients will be diagnosed and 13,120 patients will die from kidney cancer. Data from the Surveillance, Epidemiology, and End Results program of the National Cancer Institute illustrates the steady rise of RCC incidence over the past 30 years. The majority of clear cell RCC patients have mutations in the von Hippel-Lindau (VHL) gene that result in unregulated expression of the hypoxia-inducible factor (HIF) transcription factor complex. Based on this recognition of the role of hypoxia in RCC carcinogenesis, most patients with metastatic RCC are treated with agents that target the HIF axis, either in the form of a VEGF or mTOR inhibitor. The VEGF inhibitors including small molecule multi-tyrosine kinase inhibitors (TKIs) such as sunitinib, pazopanib, sorafenib, and axitinib, and antibodies to VEGF such as bevacizumab have all shown clinical activity in RCC. Surprisingly however, despite the wide availability of these agents to treat RCC, many challenges remain: VEGF expression patterns and activation of downstream pathways in RCC are not well characterized. TKIs are non-selective and often incur significant toxic off-target effects that result in subtherapeutic treatment and futile toxicity in the 20% that do not respond. Thus, there is an urgent need to identify molecular markers that identify the relevant oncogenic pathway in each tumor to aid in the selection of systemic treatments and drastically reduce the amount of futile toxicity. However, identifying and developing biomarkers in RCC has been extremely challenging and a major deterrent has been the inability to measure specific biologic proteomic alterations in patient cells before, during and after treatment. Using existing methods, it is virtually impossibl to obtain real-time proteomic information from clinical specimens. We developed nanoscale immunoassay (NIA) as a highly sensitive, quantitative and unique way to concurrently measure proteins, their different isoforms and multiple phosphorylation states. Further, nanoscale proteomic analysis has the potential to dramatically reduce the amount of tissue required for diagnosis both in primary or other metastatic site biopsy. The ability to perform proteomic analysis of multiple tumor sites at a single time point from same patient, as well as in multiple samples over time, will accelerate translational research and ultimately improve care for patients with RCC. We believe that molecular markers hold the promise to inform every step of patient care in RCC including diagnosis, selection of surgical and systemic treatment, and monitoring response to targeted therapies. We aim to use NIA to identify distinct proteomic signatures of the commonly dysregulated hypoxia pathway in RCC patients and thus aid the selection of personalized and effective systemic therapies by targeting the patient's tumor-specific pathway(s). PUBLIC HEALTH RELEVANCE: Renal cell carcinoma (RCC, kidney cancer) is one of the most commonly diagnosed cancers and is associated with significant mortality. The treatment of RCC suffers from several critical issues that can be addressed using a novel nanoscale immunoassay (NIA), which is a highly sensitive and quantitative proteomic assay, for in vivo analysis of oncoproteins in scant clinical material. Our goal is to develop and validate NIA as a platform which can analyze clinical material from primary and metastatic RCC tumors and paired normal tissues, to discover proteomic biomarkers for diagnosis, treatment selection, and measures of therapeutic response.